title: Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis.
abstract: Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
authors.forename: Yael
authors.initials: Y
authors.lastname: Shenkerman
authors.forename: Yifat
authors.initials: Y
authors.lastname: Elharar
authors.forename: Marina
authors.initials: M
authors.lastname: Vishkautzan
authors.forename: Eyal
authors.initials: E
authors.lastname: Gur
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D017353:Gene Deletion; D005798:Genes, Bacterial; D020102:Mycobacterium smegmatis / Q000235:genetics*
chemical_list: D017931:DNA Primers
keywords: 20S; Aph; Che9c; Cre; DNA sequences recognized by the Cre recombinase; GFP; Gene deletion; Homologous recombination; Hyg; Mycobacterium; P1; PafA; Pup; RFP; a Bacillus subtilis gene encoding the enzyme levansucrase; a Che9c protein encoded by the 60 gene; a Che9c protein encoded by the 61 gene; a P1 recombinase; a hygromycin resistance gene; a kanamycin resistance gene; a mycobacterial bacteriophage; an Escherichia coli bacteriophage; double stranded DNA; dsDNA; gp60; gp61; green fluorescence protein; loxP sites; prcA; prcB; prcS; prokaryotic ubiquitin like protein; proteasome accessory factor A; proteasome catalytic particle; red fluorescence protein; sacB; the Mycobacterium smegmatis gene encoding Pup; the Mycobacterium smegmatis gene encoding the proteasome α subunit; the Mycobacterium smegmatis gene encoding the proteasome β subunit